The broad goal of this proposal is to contribute to the understanding of the mechanism of blood clot dissolution, or fibrinolysis, by plasmin. Specifically we propose to complete the covalent structure determination of the heavy chain of human plasmin. Activation of human plasminogen (MW approximately 85,000) produces plasmin, a molecule consisting of a heavy chain and a light chain linked by a disulfide bond. The light chain (MW approximately 26,000) resembles trypsin, thrombin and other coagulation factors in terms of primary structure and function. Little is known of the structure and function of the heavy chain (MW approximately 55,000). Numerous similarities in gross structure and mode of action exist between plasmin and other coagulation proteins, and one can logically ask whether there are similarities in primary structure between plasmin and these or other proteins such as immunoglobulins. Also does the heavy chain contain the binding site for its substrate, fibrin, or for plasmin activators? These questons can be answered in part by determining the amino acid sequence of plasmin and comparing it with that of other proteins. We have already sequenced about 30% of the heavy chain and now have some idea of the gross structure of the molecule. We have also discovered evidence for the gene duplication in the heavy chain, similar to that recently reported for prothrombin. We now plan to finish the primary sequence and to attack the complex problem of determining the disulfide linkages (23 in all) in plasminogen. Sequence studies will be carried out using conventional methods for protein fragmentation and peptide isolation as well as some new innovations under development in our laboratory. Of particular use in these studies is the Edman degradation using the automatic solid-phase sequencer, which was developed in our laboratory. This instrument is now capable of sequencing both peptides and proteins at a rate of about 25 cycles per day. Special techniques involving convalent and affinity chromatography will be used for isolating disulfide and carbohydrate peptides.